Blood platelets provide the cellular element for hemostasis, support the endothelium and play a role in the proliferation of smooth muscle cells leading to arteriosclerosis. The objective of this proposal is to investigate the structure and function of a high molecular weight platelet glycorprotein, designated thrombospondin, which is rapidly released from a platelet granule fraction in response to thrombin. Specific focus will be directed to the following areas: (1) Structure of thrombospondin. The shape of the intact thrombospondin molecule has been determined and structural characterization can now be extended to the the subunit and supermolecular levels. Efforts will be made to separate the subunit chains of thrombosponding and to characterize them in terms of carbohydrate content, N-terminal amino acid sequence and two-dimensional peptide mapping. In addition, the hypothesis that potential sites for proteolytic cleavages are blocked by noncovalent associations or conformational changes in thrombospondin will be examined by applications of protein separation techniques under conditions which will not disrupt the structure of thrombospondin or potential complexes. (2) Thrombospondin as a multifunctional protein. A specific aim of this proposal is to characterize potential binding of thrombospondin to platelets, fibronectin, collagen, fibrinogen and fibrin in terms of binding affinity, number of binding sites and caclim dependence. In addition, the proteolytic fragments of thrombospondin which contain the binding domains will be identified. (3) Enzyme linked immunosorbent assay (ELISA). Indirect ELILSA for thrombospondin will be developed and used to (1) quantitate thrombospondin in platelets and other tissues, (2) to monitor thrombosponding during application of spearation techniques (3) to quantitate thrombospondin which is ireleased and associated with the platelet membrane and (4) to conduct a pilot study to determine whether or not blood levels of thrombospondin increase in disease states associated with in vivo platelet activation.